Bottom Line:
These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks.These findings are highly significant because they show for the first time that chronic wounds can be generated in an animal model effectively and consistently.The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue.

Affiliation: Departments of Cell Biology and Neuroscience, University of California Riverside, Riverside, California, United States of America; Bioengineering Interdepartmental Graduate Program, University of California Riverside, Riverside, California, United States of America.

ABSTRACTChronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼$25B/year in the US alone. We previously discovered that a genetically modified mouse model displays impaired healing similar to problematic wounds in humans and that sometimes the wounds become chronic. Here we show how and why these impaired wounds become chronic, describe a way whereby we can drive impaired wounds to chronicity at will and propose that the same processes are involved in chronic wound development in humans. We hypothesize that exacerbated levels of oxidative stress are critical for initiation of chronicity. We show that, very early after injury, wounds with impaired healing contain elevated levels of reactive oxygen and nitrogen species and, much like in humans, these levels increase with age. Moreover, the activity of anti-oxidant enzymes is not elevated, leading to buildup of oxidative stress in the wound environment. To induce chronicity, we exacerbated the redox imbalance by further inhibiting the antioxidant enzymes and by infecting the wounds with biofilm-forming bacteria isolated from the chronic wounds that developed naturally in these mice. These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks. These findings are highly significant because they show for the first time that chronic wounds can be generated in an animal model effectively and consistently. The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue. Furthermore, the model can lead to the understanding of other fundamental mechanisms of chronic wound development that can potentially lead to novel therapies.

pone-0109848-g002: Microscopic, biochemical and chemical markers show imbalanced redox in LIGHT−/− mice.(A) In vivo imaging of ROS was carried out using the ImageEM 1K EM-CCD camera with an optical system consisting of a 50 mm f/1.2 lens. Signals were obtained around the periphery of the wound as early as 4 hrs post-wounding in the LIGHT−/− mice and significantly higher signals captured in LIGHT−/− mice peaked at 24 hrs post-wounding. (B) Lactate measurements: An oxidized intermediate was formed when extracted lactate reacted with a probe to give fluorescence detectable at 605 nm. There was significant increase in levels of lactate accumulation in LIGHT−/− mice at 24–48 hrs post wounding. n = 6. (C) pH levels were measured using a beetrode microelectrode and micro-reference electrode. The LIGHT−/− wounds were systematically more acidic than controls. n = 25. (D,E) Methanolic-extracted nitrite (D) and nitrate (E) were analyzed. Both were greatly increased in LIGHT−/− mice during early response to wounding. n = 8. (F-G) Phospho-eNOS levels and iNOS expression in LIGHT−/− wounds were examined by western blotting (representative experiment shown). Analysis by densitometry (normalized to C57BL/6 mouse wound). Time zero represents unwounded skin except inFigure 2C. All data are Mean ± SD. *p<0.05,**p<0.01,***p<0.001.

Mentions:
In order to identify parameters in the wounds with impaired healing that, when changed, may lead these wounds to become chronic, we first characterized the state of ROS/RNS in the early stages of impaired healing by examining a variety of components of the oxidative and nitrosative stress cycle as represented schematically in Figure S1 in File S1 Superoxide dismutase (SOD) dismutates superoxide anions (O2−) to generate H2O2, which can then be detoxified by catalase to H2O+O2 and by glutathione peroxidase (GPx) to H2O. ROS can also enter the Fenton reaction in the presence of ferrous ions to give rise to.OH+OH−. O2− can also interact with nitric oxide (NO) produced by nitric oxide synthase (NOS) to give rise to peroxinitrite anion (ONOO−). The effects of oxidative and nitrosative stress are shown in terms of lipid peroxidation, DNA damage, protein modification and cell death. Secondly, we will present the data on the manipulation of the redox balance that leads to development of chronic wounds including the characterization of the polymicrobial environment that favors growth of biofilm-forming aerobic and anaerobic bacteria. For all figures (Figures 1, 2, 3, and 4) except (Figure 2C), time t = 0 represents unwounded skin.

pone-0109848-g002: Microscopic, biochemical and chemical markers show imbalanced redox in LIGHT−/− mice.(A) In vivo imaging of ROS was carried out using the ImageEM 1K EM-CCD camera with an optical system consisting of a 50 mm f/1.2 lens. Signals were obtained around the periphery of the wound as early as 4 hrs post-wounding in the LIGHT−/− mice and significantly higher signals captured in LIGHT−/− mice peaked at 24 hrs post-wounding. (B) Lactate measurements: An oxidized intermediate was formed when extracted lactate reacted with a probe to give fluorescence detectable at 605 nm. There was significant increase in levels of lactate accumulation in LIGHT−/− mice at 24–48 hrs post wounding. n = 6. (C) pH levels were measured using a beetrode microelectrode and micro-reference electrode. The LIGHT−/− wounds were systematically more acidic than controls. n = 25. (D,E) Methanolic-extracted nitrite (D) and nitrate (E) were analyzed. Both were greatly increased in LIGHT−/− mice during early response to wounding. n = 8. (F-G) Phospho-eNOS levels and iNOS expression in LIGHT−/− wounds were examined by western blotting (representative experiment shown). Analysis by densitometry (normalized to C57BL/6 mouse wound). Time zero represents unwounded skin except inFigure 2C. All data are Mean ± SD. *p<0.05,**p<0.01,***p<0.001.

Mentions:
In order to identify parameters in the wounds with impaired healing that, when changed, may lead these wounds to become chronic, we first characterized the state of ROS/RNS in the early stages of impaired healing by examining a variety of components of the oxidative and nitrosative stress cycle as represented schematically in Figure S1 in File S1 Superoxide dismutase (SOD) dismutates superoxide anions (O2−) to generate H2O2, which can then be detoxified by catalase to H2O+O2 and by glutathione peroxidase (GPx) to H2O. ROS can also enter the Fenton reaction in the presence of ferrous ions to give rise to.OH+OH−. O2− can also interact with nitric oxide (NO) produced by nitric oxide synthase (NOS) to give rise to peroxinitrite anion (ONOO−). The effects of oxidative and nitrosative stress are shown in terms of lipid peroxidation, DNA damage, protein modification and cell death. Secondly, we will present the data on the manipulation of the redox balance that leads to development of chronic wounds including the characterization of the polymicrobial environment that favors growth of biofilm-forming aerobic and anaerobic bacteria. For all figures (Figures 1, 2, 3, and 4) except (Figure 2C), time t = 0 represents unwounded skin.

Bottom Line:
These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks.These findings are highly significant because they show for the first time that chronic wounds can be generated in an animal model effectively and consistently.The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue.

Affiliation:
Departments of Cell Biology and Neuroscience, University of California Riverside, Riverside, California, United States of America; Bioengineering Interdepartmental Graduate Program, University of California Riverside, Riverside, California, United States of America.

ABSTRACTChronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼$25B/year in the US alone. We previously discovered that a genetically modified mouse model displays impaired healing similar to problematic wounds in humans and that sometimes the wounds become chronic. Here we show how and why these impaired wounds become chronic, describe a way whereby we can drive impaired wounds to chronicity at will and propose that the same processes are involved in chronic wound development in humans. We hypothesize that exacerbated levels of oxidative stress are critical for initiation of chronicity. We show that, very early after injury, wounds with impaired healing contain elevated levels of reactive oxygen and nitrogen species and, much like in humans, these levels increase with age. Moreover, the activity of anti-oxidant enzymes is not elevated, leading to buildup of oxidative stress in the wound environment. To induce chronicity, we exacerbated the redox imbalance by further inhibiting the antioxidant enzymes and by infecting the wounds with biofilm-forming bacteria isolated from the chronic wounds that developed naturally in these mice. These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks. These findings are highly significant because they show for the first time that chronic wounds can be generated in an animal model effectively and consistently. The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue. Furthermore, the model can lead to the understanding of other fundamental mechanisms of chronic wound development that can potentially lead to novel therapies.